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Investigations on Cryptodiaporthe Corni: a Pathogen of Pagoda Dogwood

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Investigations on Cryptodiaporthe Corni: a Pathogen of Pagoda Dogwood Investigations on Cryptodiaporthe corni: a Pathogen of Pagoda Dogwood A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Garrett Lee Beier IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE Advisors: Stan C. Hokanson and Robert A. Blanchette April 2013 1 © Garrett Lee Beier 2013 2 Acknowledgements There are many people I would like to acknowledge. To Dr. Stan Hokanson, thank you for urging me to pursue higher education. Your guidance and friendship is appreciated more than you know. I have learned more teaching with you than I ever could have hoped for. To Dr. Robert Blanchette, thank you for teaching me about the wonderful world of fungi. I also thank you for always having your door open when questions arose and for not giving me a straight answer, I am better for it. To the other members on my advisory committee, Dr. Jeffrey Gillman and Dr. Jennifer Juzwik, I thank you both for being a part of the planning process, I could not have done it without you. To Benjamin Held, thank you for the constant help in the lab and listening to my tales of woe. A finer lab mate would be difficult to find. To my family, I owe a great debt, you have always had faith in me and for that I am truly grateful. Mom and Dad, you taught me the meaning of hard work and without that I would have quit a long time ago. To Kara Brehm, thank you for helping me become a better person and always listening, I couldn’t have done it without you. Also, I would like to thank the Minnesota Department of Natural Resources for allowing me to work in the various state parks. Thank you to all of the people who sent me disease samples, part of my thesis could not have been done without your help. i Abstract Pagoda dogwood (Cornus alternifolia L.) is a small understory tree native to the eastern half of North America. The plant is found both in the managed landscape and growing native in rich woodlands and swampy thickets. A major problem for the tree in both the managed and natural landscape is a devastating canker disease called golden canker or Cryptodiaporthe canker of pagoda dogwood, which is caused by the Ascomycete fungus, Cryptodiaporthe corni Wehm. The devastating effects of this fungus have caused concern among gardeners and arborists. This thesis reports results on a variety of different aspects of the host/pathogen interaction and include whether C. corni is capable of living as an endophyte in pagoda dogwood, more information about the etiology and epidemiology of golden canker, description of an effective artificial inoculation protocol resulting in disease, and the proper taxonomic placement of C. corni based on phylogenetic analysis. ii Table of Contents Page Acknowledgements i Abstract ii Table of Contents iii List of Tables iv List of Figures vi Chapter 1. Introduction Pagoda Dogwood (Cornus alternifolia L.) 1 Golden Canker of Pagoda Dogwood 1 Cryptodiaporthe corni Wehm. 3 Objectives 4 Chapter 2. Cryptodiaporthe corni Wehm., a Latent Pathogen in Asymptomatic Stems of Pagoda Dogwood (Cornus alternifolia L.) Introduction 5 Materials and Methods 8 Results 11 Discussion 13 Tables and Figures 18 Chapter 3. Inoculation of Cornus alternifolia L. with Cryptodiaporthe corni Wehm. and Etiology of the Disease Introduction 24 Materials and Methods 26 Results 32 Discussion 36 Tables and Figures 44 Chapter 4. Optimal Temperature for Growth and Toxin Production of Cryptodiaporthe corni Wehm. Introduction 51 Materials and Methods 52 Results 56 Discussion 58 Tables and Figures 62 Chapter 5. Genetic Diversity of Cryptodiaporthe corni Wehm. and Placement Within the Cryphonectriaceae Family Introduction 66 Materials and Methods 68 Results 72 Discussion 74 Tables and Figures 76 Bibliography 85 iii List of Tables Page Table 2.1. Fungicide applications on the large nursery pagoda 18 dogwoods for the 6 months prior to collection of the stems. Stems were later sampled for presence of C. corni. Table 3.1. Description and geographical origin of isolates of C. corni 44 collected from pagoda dogwood used in this study. Table 3.2. Greenhouse Inoculation Experiment 45 Observations on pagoda dogwood stems 7 months post inoculation for four different treatments. Observations are given as a proportion of stems with a (+) scoring. Inoculated plants were inoculated with C. corni growing on malt extract agar, while mock-inoculated plants were inoculated with sterile grain seed. The main stem was severed at approximately 46 cm and inoculum was placed on the wound. Table 3.3. Greenhouse Inoculation Experiment 46 Effects of 5 different C. corni isolates on variables observed on pagoda dogwood stems 3 and 7 months post inoculation (MPI) (wilt treatment and extended drought treatment were combined for analysis). Observations are given as a proportion of stems with a (+) scoring. The main stem was severed at approximately 46 cm and inoculum was placed on the wound. Table 3.4. Greenhouse Inoculation Experiment 47 Observations on pagoda dogwood stems 7 months post inoculation for four different treatments. Measurements are giving as a mean±SD and observations are given as a proportion of stems with a (+) scoring. Inoculated plants were inoculated with C. corni growing on malt extract agar, while mock-inoculated plants were inoculated with sterile grain seed. The main stem was wounded 15 cm above the soil line with a 5 mm cork borer and inoculum was placed on the wound. Table 3.5. Greenhouse Inoculation Experiment 48 Effects of 5 different C. corni isolates on variables observed on pagoda dogwood stems 3 and 7 months post inoculation (MPI) (wilt treatment and extended drought treatment were combined for analysis). Measurements are given as a mean±SD and observations are given as a proportion of stems with a (+) scoring. The main stem was wounded 15 cm above the soil line with a 5 mm cork borer and inoculum was placed on the wound. iv Table 3.6. Field Inoculation Experiment 49 Measurements and observations on pagoda dogwood branches 3 months post inoculation (MPI) for branches severed either above or below a node to make a wound. Two isolates of C. corni were used as inoculum (MNS1005 and MNS1007) for the inoculated branches, and sterilized grain seeds were used for the mock- inoculated. Measurements are given as a mean percentage and observations are given as a proportion of branches with a (+) scoring. Table 3.7. Field Inoculation Experiment 50 Measurements and observations on pagoda dogwood branches 3 months post inoculation (MPI) for branches wounded with a 5 mm cork borer. Two isolates of C. corni were used as inoculum (MNS1005 and MNS1007) for the inoculated branches, and sterilized grain seeds were used for the mock-inoculated branches. Measurements are given as a mean±SD and observations are given as a proportion of branches with a (+) scoring. Table 4.1. Proportions of pagoda dogwood stems with wilted leaves 65 (+) after being immersed in four different liquid media treatments. Inoculated media contained fungal exudates of C. corni. Observations were taken at 6 different time points. Stems with any leaves wilted were scored as a (+). Table 5.1. Description and geographical origin of isolates of C. corni 76 collected from pagoda dogwood used in this study. Table 5.2. Statistical summary for phylogenetic analysis of the four 77 different gene regions. v List of Figures Page Figure 2.1. Illustration of stem processing of pagoda dogwood from 19 initial cutting to final plating of stem tissue onto acidified malt extract agar for C. corni isolation. Figure 2.2. Appearance of C. corni on malt extract agar before (left) 20 and after (right) 3% KOH staining. This reagent provides a diagnostic test for fungi in the Cryphonectriaceae family, which causes orange stromatic tissue to turn purple. Figure 2.3. Illustration showing presence or absence of C. corni 21 throughout asymptomatic stems of pagoda dogwood collected in April 2011 (Collection 1) from 5 sites across Minnesota. Figure 2.4. Illustration showing presence or absence of C. corni 22 throughout asymptomatic stems of pagoda dogwood collected in July 2011 (Collection 2) from 5 sites across Minnesota. Figure 2.5. Increase in odds ratio (odds of finding presence of C. corni) 23 based on an increase in segment diameter (mm) within asymptomatic stems of pagoda dogwood collected from five sites across Minnesota. Dotted lines represent 95% confidence intervals. Figure 4.1. Boxplots of growth of C. corni (isolates MNS1005 and 62 MNS1006) at eight different temperature regimes (5 – 40 °C) for 3, 6, 9, and 11 days after being placed on malt extract agar. Figure 4.2. Number of stems of pagoda dogwood with wilted leaves 63 (+) after being placed in one of the four different treatments at 6 given time points after immersion in the liquid media. Inoculated media contained fungal exudates of C. corni. Stems containing a single leaf that was wilted were scored as (+). Figure 4.3. Comparison of non-wilted (-) (left) to wilted (+) (right) 64 leaves of pagoda dogwood after 52 hours in non-autoclaved mock- inoculated liquid media (left) and non-autoclaved inoculated liquid media (right). Inoculated media contained fungal exudates of C. corni. vi Figure 5.1. Neighbor-joining tree created from Beta tubulin 1 78 gene sequences to compare isolates of Cryptodiaporthe corni. Confidence levels above 50% based on 1000 bootstrap replications are listed for maximum parsimony (top) and neighbor-joining (bottom). The species for sequences obtained from Genbank are followed by the strain. Two isolates of Diaporthe ambigua were used as an outgroup. Figure 5.2. Neighbor-joining tree created from Beta tubulin 2 gene 79 sequences to compare isolates of Cryptodiaporthe corni.
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